Proper control of gene expression is important for nearly all biological processes, ranging from development to oncogenesis. One important mechanism for controlling gene expression is post-transcriptional regulation, which is normally mediated through specific sequences in the messenger RNA (mRNA) itself and often results in changes in mRNA stability and/or modulation of translational initiation. One prominent post-transcriptional regulatory mechanism is that mediated by microRNAs (miRNAs). These small RNAs recognize complementary sequences in a target mRNA and repress gene expression, primarily through mRNA destabilization as well as by inhibition of translation initiation. Nevertheless, the molecular mechanism by which miRNAs mediate repression of translation initiation is unclear. More broadly, how the translation initiation machinery binds mRNAs on a transcriptome-wide scale and the extent to which such interactions are regulated remain uncharacterized. I have had a long-standing interest in post-transcriptional regulatory mechanisms. During my graduate studies at the University of Oxford with Dr. Chris Norbury, which were funded by a Rhodes Scholarship, I uncovered an unknown mRNA decay pathway in Schizosaccharomyces pombe. Then, as a post-doctoral fellow in the laboratory of Dr. David Bartel at the Whitehead Institute, I continued to investigate RNA decay pathways and studied the degradation of miRNAs. Although the majority of miRNAs are stable, I identified several members of the extended miR-16 family as unusually unstable, and furthermore showed that this instability enabled dynamic regulation of the family in the cell cycle. This work was funded by a Ruth L. Kirschstein NRSA fellowship. My long-term goal is to understand interactions between mRNAs and regulatory factors and to define how these interactions in turn modulate gene expression on a transcriptome-wide scale. I would like to pursue this exciting topic as the leader of a research group in an academic institution. To achieve this goal, the overall objectives of this application are: 1) to identify the step(s) in translation initiation that are modulated by regulatory processes; 2) to receive training in additional scientific skills, especialy in the area of computational biology, so that I can be successful as an independent investigator. The rationale that underlies the proposed research is that transcriptome-wide mapping of the binding sites of translation initiation factors will provide insight into regulatory mechanisms and will simultaneously enable me to develop computational skills. The work proposed here comprises two aims. In Aim 1, which will be completed during the mentored phase, I will investigate the molecular mechanism(s) by which miRNAs repress translation initiation, a topic that remains unknown and controversial. To test my central hypothesis that the miRNA-mediated translational repression acts by disrupting eIF4G binding, I will map eIF4G-mRNA interactions transcriptome-wide and determine how these interactions are affected by miRNAs. A novel approach to this important issue, the proposed work will assay effects of miRNA-mediated repression on a level formerly restricted to the mechanistic studies but on the scale of the transcriptome-wide approaches. Importantly, this aim will allow me to develop experimental and computational skills for studying protein-RNA interactions transcriptome-wide in the context of translation. With its long experience in miRNA biology, deep computational expertise and consistent technological innovation, the Bartel laboratory is the ideal place for this training. In Aim 2, which I will initiate during the mentored phase and complete in the independent phase, I will extend this work to map binding of other initiation factors on a transcriptome-wide scale. Hypothesizing that regulation often occurs through differential, controlled binding of initiation factors, I will map the binding of other factors, such as eIF4E and eIF4A, and determine how these interactions relate to translational efficiency. I will also apply these analyses to naturall non-translated transcripts, cytoplasmic long intervening non-coding RNAs (lincRNAs), and thereby shed light on specific mechanisms abrogating translation. Once integrated, these results will provide a new perspective from which to consider post-transcriptional gene regulation. Through my previous work, I have extensive molecular biology training as well as experience in high- throughput sequencing and basic computational analysis. My experimental background and expertise in post- transcriptional regulation ideally positions me for the proposed work. The training and research described in this proposal will allow me to develop a tool-kit of approaches that will form the basis of my independent research, and will provide the platform from which to launch my career as an independent investigator. PUBLIC HEALTH RELEVANCE: Although all cells have the same collection of genes, what makes them different is which genes are turned on and which are turned off. When genes are inappropriately expressed, cancer can sometimes develop and/or the development of an organism can occur improperly. This proposal focuses on comprehensively understanding how the final step in turning on a gene,-that is, making the protein product,-is controlled.